FIGURE 6.

NIK is essential for optimum induction of a set of TNF-induced late responsive gene expression. A, iKO simulation of NIK is shown. The n3a parameter represents the NIK transcription rate in the system, which plays an important role in feed-forward signaling. A simulation of p52 processing and subsequent non-canonical signaling was performed by in silico knockdown of transcription of NIK by making n3a = 0.0. B, á Western blot shows NIK knockdown in A549 cells by 50 and 100 nm NIK-specific siRNA as compared with the cells transfected with nonspecific control siRNA. β-Actin in the lower panel serves as a loading control. C–F, A549 cells transfected with control (dark bars) or NIK specific siRNA (light bars) were treated with TNF (25 ng/ml) for various time intervals. RNA isolated thereafter was used to quantify expression of A20, IL8, Naf1, and NF-κB2 by Q-RT-PCR using specific primers. Data are expressed as -fold change as compared with untransfected cells after normalizing to internal controls, GAPDH. Data represent the mean ± S.D. of three independent experiments and analyzed by two-way ANOVA with multiple comparison (time and siRNA treatment) and Tukey's post hoc test for significance between time intervals and the siRNA- treatment groups. Significantly different from TNF (0 h)-treated samples: *, p < 0.05; **, p < 0.001; significantly different from control siRNA-transfected samples: †, p < 0.01.